Method for generating human multipotent mammary stem cells from normal primary breast luminal cells

Abstract

The invention relates to methods for generating multipotent mammary stem cells from isolated and cultured human breast luminal cells. The method comprises the steps: 1. isolating and growing normal differentiated cells in vitro; 2. treating the differentiated cells with either a conditioned medium from active fibroblasts or cytokine. The invention also relates to multipotent mammary stem cells, cultures of the multipotent stem cells, differentiated cells, tissues, organs derived from the culture multipotent stem cells isolated by the methods disclosed and therapeutic and other uses for those cells thereof.

We claim: 
     
       1. A method of tissue engineering, transplantation, tissue reconstruction, cell therapy procedure, or treatment of the effects of mastectomy, comprising the steps:
 A) generating multipotent mammary stem cells by: 
 a) identifying and isolating primary breast luminal cells, and growing the identified and isolated primary breast luminal cells in-vitro, 
 b) treating the primary breast luminal cells with either a conditioned medium from active breast stromal fibroblasts or with interleukin-8 (IL-8) in serum-free medium; 
 c) continuing the growth of the treated cells until the formation of cellular aggregates, wherein growth of the cells is performed in a breast luminal cell-specific medium, wherein said breast luminal cell-specific medium comprises a 1:1 mixture (vol/vol) of DMEM medium and F12 medium supplemented with 1%-20% (vol/vol) serum, and supplemented with 0.1%-2% (vol/vol) of an antibiotic-antimycotic solution, said antibiotic-antimycotic solution comprising 10,000 units/ml penicillin, 10,000 μg/ml streptomycin and 25 μg/ml amphotericin B; 
 d) transferring the cells to a stem cell growth medium and growing them further until the formation of mammospheres, wherein said stem cell growth medium comprises mammary epithelial basal medium (MEBM) supplemented with 0.1%-2% (vol/vol) of an antibiotic-antimycotic solution, said antibiotic-antimycotic solution comprising 10,000 units/ml penicillin, 10,000 μg/ml streptomycin and 25 μg/ml amphotericin B; and 
 e) isolating said multipotent mammary stem cells; and 
 B) applying/administering said multipotent mammary stem cells to a patient in need thereof at a place of the patient's body w here a process of tissue engineering, transplantation, tissue reconstruction or cell therapy procedure is desired. 
 
     
     
       2. The method according to  claim 1 , wherein the conditioned medium from active breast stromal fibroblasts of step b) comprises a 1:1 mixture (vol/vol) of M199 medium and F12 medium, supplemented with 0%-1% (vol/vol) serum, and 0.1%-2% (vol/vol) of an antibiotic-antimycotic solution, said antibiotic-antimycotic solution comprising 10,000 units/ml penicillin, 10000 μg/ml streptomycin and 25 μg/ml amphotericin B. 
     
     
       3. The method according to  claim 1 , wherein treating in step b) is done for a period in the range of from 12 hours to 36 hours. 
     
     
       4. The method according to  claim 1 , wherein the primary breast luminal cells are primary human breast luminal cells, and the multipotent mammary stem cells are multipotent human mammary stem cells. 
     
     
       5. The method according to  claim 1 , wherein said interleukin-8 is recombinant interleukin-8. 
     
     
       6. The method according to  claim 1 , wherein said interleukin-8 is human interleukin-8 having an amino acid sequence represented by 
       
         
           
                 
               
                   (SEQ ID NO: 1) 
                 
                   MTSKLAVALLAAFLISAALCEGAVLPRSAKELRCQCIKTYSKPFHPKFIK 
                 
                   ELRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRAENS. 
                 
             
                
                
                
               
            
           
         
       
     
     
       7. The method according to  claim 1 , wherein said interleukin-8 is used in step b) at a concentration in the range of from 25 ng/mL to 100 ng/mL. 
     
     
       8. The method according to  claim 1 , wherein said primary breast luminal cells are identified and isolated based on their expressing at least one or a combination of markers selected from Cytokeratin 18, Cytokeratin 19 and epithelial cell adhesion molecule (EpCam), wherein they are selected and isolated if they express such marker(s), and are not selected and isolated if they do not, and/or wherein said primary breast luminal cells are identified and isolated based on their not expressing at least one or a combination of the following markers selected from Cytokeratin 14 and integrin α-6 (CD49f), wherein they are selected and isolated if they do not express such marker(s), and are not selected and not isolated if they do. 
     
     
       9. The method according to  claim 1 , wherein the growing of said cells in step d) occurs in plates or dishes with a low attachment substrate. 
     
     
       10. The method according to  claim 1 , wherein the formed mammospheres have a size in the range of from 50 μm to 200 μm. 
     
     
       11. The method according to  claim 1 , wherein the multipotent mammary stem cells are produced by the performance of steps a)-d) and are characterized by an up-regulation of one or several or all of CD44, ALDH1, ALDH2, OCT4, KLF4 and N-cadherin, and the simultaneous expression of one or several or all myoepithelial and luminal markers selected from CyK5, CyK6, CyK14, CyK18, CyK19, c-Kit, Elf-5, in comparison to primary breast luminal cells not having undergone a treatment of steps a)-d), and are further characterized by a lower expression of CD24 and E-cadherin in comparison to primary breast luminal cells not having undergone a treatment of steps a)-d). 
     
     
       12. The method according to  claim 1 , which is a cosmetic method or which is a medical treatment method. 
     
     
       13. A method of tissue engineering, transplantation, tissue reconstruction, cell therapy procedure, or treatment of the effects of mastectomy, comprising the steps:
 A) generating multipotent mammary stem cells by: 
 a) identifying and isolating primary breast luminal cells, and growing the identified and isolated primary breast luminal cells in-vitro; 
 b) treating the primary breast luminal cells with a conditioned medium from active breast stromal fibroblasts, w herein the conditioned medium from active breast stromal fibroblasts comprises a 1:1 mixture (vol/vol) of M199 medium and F12 medium, supplemented with 0%-1% (vol/vol) serum, and 0.1%-2% (vol/vol) of an antibiotic-antimycotic solution, said antibiotic-antimycotic solution comprising 10,000 units/ml penicillin, 10,000 μg/ml streptomycin and 25 μg/ml amphotericin B; 
 c) continuing the growth of the treated cells until the formation of cellular aggregates; 
 d) transferring the cells to a stem cell growth medium and growing them further until the formation of mammospheres, wherein said stem cell growth medium comprises mammary epithelial basal medium (MEBM) supplemented with 0.1%-2% (vol/vol) of an antibiotic-antimycotic solution, said antibiotic-antimycotic solution comprising 10,000 units/ml penicillin, 10,000 μg/ml streptomycin and 25 μg/ml amphotericin B; and 
 e) isolating said multipotent mammary stem cells; and 
 B) applying/administering said multipotent mammary stem cells to a patient in need thereof at a place of the patient's body w here a process of tissue engineering, transplantation, tissue reconstruction or cell therapy procedure is desired. 
 
     
     
       14. The method according to  claim 13 , wherein treating in step b) is done for a period of from 12 hours to 36 hours. 
     
     
       15. The method according to  claim 13 , wherein the primary breast luminal cells are primary human breast luminal cells, and the multipotent mammary stem cells are multipotent human mammary stem cells. 
     
     
       16. A method of tissue engineering, transplantation, tissue reconstruction, cell therapy procedure, or treatment of the effects of mastectomy, comprising the steps:
 A) generating multipotent mammary stem cells by: 
 a) identifying and isolating primary breast luminal cells, and growing the identified and isolated primary breast luminal cells in-vitro; 
 b) treating the primary breast luminal cells with either a conditioned medium from active breast stromal fibroblasts or with interleukin-8 (IL-8) in serum-free medium; 
 c) continuing the growth of the treated cells until the formation of cellular aggregates; 
 d) transferring the cells to a stem cell growth medium and growing them further until the formation of mammospheres, wherein said stem cell growth medium comprises mammary epithelial basal medium (MEBM), supplemented with 0.1%-2% (vol/vol) of an antibiotic-antimycotic solution, said antibiotic-antimycotic solution comprising 10,000 units/ml penicillin, 10,000 μg/ml streptomycin and 25 μg/ml amphotericin B; and 
 e) isolating said multipotent mammary stem cells; 
 and 
 B) applying/administering said multipotent mammary stem cells to a patient in need thereof at a place of the patient's body w here a process of tissue engineering, transplantation, tissue reconstruction or cell therapy procedure is desired. 
 
     
     
       17. The method according to  claim 16 , wherein said interleukin-8 is recombinant interleukin-8. 
     
     
       18. The method according to  claim 16 , wherein said interleukin-8 is human interleukin-8 having an amino acid sequence represented by 
       
         
           
                 
               
                   (SEQ ID NO: 1) 
                 
                   MTSKLAVALLAAFLISAALCEGAVLPRSAKELRCQCIKTYSKPFHPKFIKE 
                 
                     
                 
                   LRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRAENS. 
                 
             
                
                
                
                
               
            
           
         
       
     
     
       19. The method according to  claim 16 , wherein said interleukin-8 is used in step b) at a concentration in the range of from 25 ng/mL to 100 ng/mL. 
     
     
       20. The method according to  claim 16 , wherein said stem cell growth medium consists of mammary epithelial basal medium (MEBM), supplemented with 0.1%-2% (vol/vol) of an antibiotic-antimycotic solution, said antibiotic-antimycotic solution comprising 10,000 units/ml penicillin, 10000 μg/ml streptomycin and 25 μg/ml amphotericin B.